https://onlinelibrary.wiley.com/doi/pdf/10.1002/jor.1100090504

Mesenchymal stem cells

In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells

Osteoblast adhesion on biomaterials.

MSCs are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are rare in bone marrow, representing approximately 1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo. The current review describes what is known about MSCs and their capacity to home to tissues together with the associated molecular mechanisms involving chemokine receptors and adhesion molecules.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/epdf/10.1634/stemcells.2007-0197

Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro

When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.

Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo.

The effect of parathyroid extract on cellular activity and plasma calcium levels in vivo.

Osteogenic tissue formed in vivo in diffusion chambers inoculated with a suspension of marrow cells contains both bone and cartilage. Observations by transmission electron microscopy on the transitional zone between these tissues showed a mixture of osseous and cartilaginous features in both matrix and cells. Two forms of collagen, morphologically consistent with Types I and II, are found in intimate association within the same intercellular septa. The results suggest the possibility that the different collagen types are synthesized by the same cells and that the variation in cellular morphology could be associated with the changes in the type of collagen synthesized. This unique characteristic of calcified tissues formed in diffusion chambers is probably the result of isolation from direct blood circulation, mechanical stress, and cellular mechanisms of tissue breakdown.

Ultrastructure of bone and cartilage formed in vivo in diffusion chambers.

Localized regions of mineralization were found in confluent cultures of rabbit marrow fibroblastic cells. The mineralized tissue developed within clusters of giant fat cells in the spaces between the cells. Investigations with light and electron microscopy demonstrated that in these sites there was some differentiation of the fibroblastic cells in an osteogenic direction, shown by changes to more polygonal shapes, and the synthesis of well-banded collagen similar to that found in bone tissue. Differentiation may be due, in part, to increased cell density in a confined space. Growth of the mineralized tissue was observed in the living cultures with a fluorescence microscope. Electron probe microanalysis confirmed that the mineral formed was hydroxyapatite. Initiating sites of mineralization included membranous vesicular bodies, lipid, and products of cellular degeneration. Once initiated, mineralization appeared to spread rapidly into adjacent collagenous and other structures, suggesting the appearance of a mixture of skeletal-type and dystrophic mineralization.

Maureen Owen

Papers

Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo.

The effect of parathyroid extract on cellular activity and plasma calcium levels in vivo.

Ultrastructure of bone and cartilage formed in vivo in diffusion chambers.

Mineralization in in vitro cultures of rabbit marrow stromal cells.

The osteogenic capacity of bone narrow cells.